Changes in β-Adrenergic Receptor Subtypes in Alzheimer-Type Dementia

Using ligand binding techniques, we studied beta-adrenergic receptor subtypes in brains obtained at autopsy from seven histologically normal controls and seven histopathologically verified cases with Alzheimer-type dementia (ATD). Inhibition of [3H]dihydroalprenolol [( 3H]DHA) binding by the selective beta 1 antagonist, metoprolol, results in nonlinear Hofstee plots, suggesting the presence of the two receptor subtypes in the human brain. The calculated ratios of beta 1/beta 2-adrenergic receptors in control brains are as follows: frontal cortex, 49:51; temporal cortex, 31:69; hippocampus, 66:34; thalamus, 23:77; putamen, 70:30; caudate, 48:52; nucleus basalis of Meynert (NbM), 43:57; cerebellar hemisphere, 25:75. Compared with the controls, total concentrations of beta-adrenergic receptors were significantly reduced only in the thalamus of the ATD brains. beta 1-Adrenergic receptor concentrations were significantly reduced in the hippocampus and increased in the NbM and cerebellar hemisphere, whereas beta 2-adrenergic receptor concentrations were significantly reduced in the thalamus, NbM, and cerebellar hemisphere and increased in the hippocampus and putamen of the ATD brains. These results suggest that beta 1- and beta 2-adrenergic receptors are present in the human brain and that there are significant changes in both receptor subtypes in selected brain regions in patients with ATD.     

Characterization and Regional Distribution of α2-Adrenoceptors in Postmortem Human Brain Using the Full Agonist [3H]UK 14304

The full agonist [3H]UK 14304 [5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline] was used to characterize alpha 2-adrenoceptors in postmortem human brain. The binding at 25 degrees C was rapid (t1/2, 4.6 min) and reversible (t1/2, 14.1 min), and the KD determined from the kinetic studies was 0.48 nM. In frontal cortex, the rank order of potency of adrenergic drugs competing with [3H]UK 14304 or [3H]clonidine showed the specificity for an alpha 2A-adrenoceptor: UK 14304 approximately equal to yohimbine approximately equal to oxymetazoline approximately equal to clonidine greater than phentolamine approximately equal to (-)-adrenaline greater than idazoxan approximately equal to (-)-noradrenaline greater than phenylephrine greater than (+/-)-adrenaline much greater than corynanthine greater than prazosin much greater than (+/-)-propranolol. GTP induced a threefold decrease in the affinity of [3H]UK 14304, with no alteration in the maximum number of binding sites, suggesting that the radioligand labelled the high-affinity state of the alpha 2-adrenoceptor. In the frontal cortex, analyses of saturation curves indicated the existence of a single population of noninteracting sites for [3H]UK 14304 (KD = 0.35 +/- 0.13 nM; Bmax = 74 +/- 9 fmol/mg of protein). In other brain regions (hypothalamus, hippocampus, cerebellum, brainstem, caudate nucleus, and amygdala) the Bmax ranged from 68 +/- 7 to 28 +/- 4 fmol/mg of protein. No significant changes in the KD values were found in the different regions examined. The binding of [3H]UK 14304 was not affected by age, sex or postmortem delay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

Properties of [3H]Prazosin-Labeled α1Adrenergic Receptors in Rat Brain and Porcine Neurointermediate Lobe Tissue

Abstract: [³H]Prazosin binding to α₁ receptors in homogenates of rat prefrontal cortical tissue and porcine pituitary neurointermediate lobe tissue was investigated. Competition curves produced by coincubating adrenergic agonists and antagonists with 0.5 n M [³H]prazosin and tissue revealed some anomalous binding properties. In the brain and pituitary tissue, agonist competition curves produced "shallow" slopes, with Hill coefficients significantly lower than unity. The IC₅₀ of the agonists epinephrine, norepinephrine, and clonidine for inhibition of 0.5 n M [³H]prazosin binding were significantly lower in the porcine pituitary than in the rat brain. Most antagonists, such as prazosin, chlorpromazine, and piperoxan, produced "steep" competition curves with Hill coefficients close to unity, with two notable exceptions. WB-4101 and phentolamine produced competition curves with Hill coefficients significantly less than unity in the rat brain preparation. Ketanserin, an antagonist, displayed a sevenfold higher affinity for the a, sites in the pituitary tissue than in the brain tissue. These anomalies in the binding results may indicate the presence of an endogenous modulatory factor affecting agonist and antagonist affinities for the a, receptor.     

Regional Distribution of α2A-and α2B-Adrenoceptor Subtypes in Postmortem Human Brain

The newly available and highly selective radiolabeled antagonist [3H]RX 821002 was used to examine the distribution of alpha 2 adrenoceptors in human brain. High densities of alpha 2 adrenoceptors were found in the hippocampus, frontal cortex, thalamus, amygdala, pons, and medulla oblongata. Intermediate densities were observed in the striatum (nucleus accumbens, nucleus caudatus, and putamen), globus pallidus, and substantia nigra. The KD values for [3H]RX 821002 were similar in all regions (ranging from 2.8 to 7.5 nM). On the basis of their different affinities for prazosin and oxymetazoline, the alpha 2 adrenoceptors have been divided into alpha 2A and alpha 2B subtypes. To examine the alpha 2A/alpha 2B-adrenoceptor ratio in the different brain regions, we performed oxymetazoline and prazosin/[3H]RX 821002 competition binding experiments. In frontal cortex membranes, the competition curves with prazosin were steep, indicating a single class of binding sites, whereas the competition curves with oxymetazoline were shallow and fitted by computer best to a two-site model. However, in the presence of GTP, the high-affinity sites for oxymetazoline were partially converted into low-affinity sites, indicating that this agonist interacts with high- and low-affinity states of the alpha 2 adrenoceptors. This implies that oxymetazoline is not very suitable for discriminating the alpha 2A- and alpha 2B-receptor subtypes in radioligand binding studies. Therefore, prazosin/[3H]RX 821002 competition binding experiments were used to investigate the distribution of the alpha 2-adrenoceptor subtypes in human brain. The alpha 2A-receptor subtype was detected in all brain regions examined. In contrast, alpha 2B receptors were only observed in striatum and globus pallidus.     

Thermodynamic Parameters of Frog Brain κ-Opioid Receptors

Abstract: The thermodynamic parameters for [³H]-ethylketocyclazocine binding in frog ( Rana esculenta ) brain membranes have been examined. Computer-based nonlinear regression analysis of the untransformed equilibrium displacement data showed that this ligand bound to two sites with different affinities and capacities in this tissue. K _A values derived from equilibrium displacement curves have been used for calculating the changes in the standard Gibbs energy, enthalpy, and entropy during the binding process. Van't Hoff plots are bipartite, with transitions occurring at 18°C for both the high- and the low-affinity sites. For the high-affinity site, the reaction appears to be associated with a decrease in enthalpy below the transition temperature and a significant gain in entropy above this temperature. The reverse appears to be true for the low-affinity site. We conclude that this profile fairly approximates the mixed agonist-antagonist nature of this ligand and surmise that thermodynamic analysis could be a very useful tool for characterization of the nature of cloned opioid receptors in vitro.     

Changes in Nicotinic and Muscarinic Cholinergic Receptors in Alzheimer-Type Dementia

Nicotinic and muscarinic cholinergic receptors were studied in autopsied brains from four histologically normal controls and five histopathologically verified cases of Alzheimer-type dementia (ATD), using ligand binding techniques. Nicotinic and muscarinic cholinergic receptors were assessed by (-)-[3H]nicotine and [3H]quinuclidinyl benzilate [( 3H]QNB), respectively. Compared with the controls, (-)-[3H]nicotine binding sites in the ATD brain regions examined were significantly reduced in the putamen and the nucleus basalis of Meynert (NbM). [3H]QNB binding was significantly reduced in the hippocampus and NbM. These findings suggest that there are significant changes of nicotinic and muscarinic cholinergic receptors in selected regions of ATD brains.     

Biochemical Characterization of the Nicotinic Cholinergic Receptors in Human Brain: Binding of (—)-[3H]Nicotine

(-)-[3H]Nicotine was found to bind specifically to membranes of human brains obtained at autopsy. The binding was stereospecific, (-)-nicotine being 40 times more potent than (+)-nicotine in displacing labeled (-)-nicotine. Saturation binding studies revealed the presence of two binding sites with dissociation constant (KD) values of 8.1 and 86 nM, and maximum binding capacity (Bmax) values of 36 and 90 fmol/mg protein, respectively. In competition studies, nicotinic agonists were 1,000 times more potent than ganglionic, neuromuscular, and muscarinic blocking drugs in displacing labeled (-)-nicotine. IC50 values for cholinergic drugs of (-)-[3H]nicotine binding were as follows: (-)-nicotine, 0.51 nM; acetylcholine, 12.6 nM; (+)-nicotine, 19.9 nM; cytisine, 27.3 nM; and carbachol, 527 nM. IC50 values of alpha-bungarotoxin, hexamethonium, d-tubocurarine, and atropine were larger than 50 microM. (-)-[3H]Nicotine binding was highest in the nucleus basalis of Meynert and thalamus and lowest in the cerebral cortex and caudate in the brain regions tested. These results suggest that nicotinic cholinergic receptors are present in human brain and that there are regional differences in the density of these receptors.     

Solubilization and Characterization of Rat Brain α2-Adrenergic Receptor

alpha 2-Adrenergic receptors labelled by [3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound alpha 2-adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level.     

Decreased Density of Presynaptic α2-Adrenoceptors in Postmortem Brains of Patients with Alzheimer''s Disease

The full agonist [3H]bromoxidine (UK 14304) was used to quantitate alpha 2-adrenoceptors in postmortem brains of patients with Alzheimer's disease. The effects of aging and human serum Cohn fraction IV on [3H]bromoxidine binding were also assessed. In patients with Alzheimer's disease, the binding capacity (Bmax) of [3H]bromoxidine was lower in the frontal cortex (37%), hypothalamus (33%), and cerebellum (52%) than in matched controls. In the hippocampus, amygdala, and head of caudate, the binding capacities (Bmax) were unchanged. Quantitative autoradiographic analyses with [3H]bromoxidine confirmed the existence of a marked reduction (55-60%) in alpha 2A-adrenoceptor density in the frontal cortex (layers I and III). In patients with dementia who did not meet neuropathological criteria for Alzheimer's disease, the density of alpha 2-adrenoceptors was unchanged. In control subjects, the density of alpha 2A-adrenoceptors in the frontal cortex showed a significant negative correlation with age at death. The inhibitory effect of human serum Cohn fraction IV on [3H]bromoxidine was very similar in control subjects and patients with Alzheimer's disease. The observed decrease in the density of brain alpha 2-adrenoceptors in Alzheimer's disease may represent direct biochemical evidence of a presynaptic location of this receptor on noradrenergic nerve terminals in the human CNS.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

Synergistic Interactions Between α1- and α2-Adrenergic Receptors in Activating 3H-Inositol Phosphate Formation in Primary Glial Cell Cultures

Norepinephrine (NE)-stimulated 3H-inositol phosphate (3H-InsP) formation in primary glial cell cultures is thought to be due to alpha 1-adrenergic receptor activation. Surprisingly, the alpha 1-selective agonists phenylephrine and methoxamine showed only 12-21% of the intrinsic activity of NE in activating this response. Although the alpha 2-selective agonist UK 14,304 was itself inactive, inclusion of UK 14,304 increased the response to the alpha 1-selective agonists by about threefold. This increase was concentration-dependent and occurred at all time points examined. 6-Fluoro-NE and alpha-methyl-NE mimicked the effect of NE in glial cultures, although with lower potencies. However, several partial agonists were ineffective in activating this response, in both the presence and absence of UK 14,304. Synergistic interactions were not observed for alpha 1-mediated responses in slices of rat cerebral cortex, either for formation of 3H-InsPs or potentiation of isoproterenol- or adenosine-stimulated cyclic AMP accumulation. Both UK 14,304 and phenylephrine inhibited NE-stimulated 3H-InsP formation in concentrations similar to those necessary to activate this response directly. These results suggest that NE activates 3H-InsP formation in primary glial cultures by synergistic actions on both alpha 1- and alpha 2-adrenergic receptors. The agonists UK 14,304 and phenylephrine also can act to inhibit the response to NE competitively.     

Phorbol Esters Mimic α-Adrenergic Potentiation of Serotonin N-Acetyltransferase Induction in the Rat Pineal

alpha-Adrenergic stimulation of the rat pineal gland is known to stimulate phosphatidylinositol turnover and to potentiate the induction of serotonin N-acetyltransferase (SNAT) activity evoked by submaximal beta-adrenergic stimulation. In some (other) systems tumor-promoting phorbol esters are known to mimic physiologic stimulation and to enhance specifically the activity of protein kinase C. Here it is shown that phorbol esters specifically mimic the potentiating effect of alpha-adrenergic stimulation on SNAT activity in the rat pineal. These effects contribute to the argument for a role for phosphatidylinositol turnover and protein kinase C in mediating alpha-adrenergic stimulation.     

Down-Regulation of β-Adrenergic Receptors by Pindolol in Gsα-Transfected S49 cyc− Murine Lymphoma Cells

The role of the alpha subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (GS alpha) in the down-regulation of beta-adrenergic receptors by pindolol was studied in S49 cyc- cells (normally GS alpha-deficient) transfected to express functional recombinant rat GS alpha. An inducible cell line (S49 GS alpha IND) was derived from S49 cyc- cells transfected with a vector containing the full-length coding sequence of GS alpha under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. GS alpha was not detectable in S49 GS alpha IND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [alpha-32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, GS alpha was present at a level 40-50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [alpha-32P]ADP-ribosylation. Membranes prepared from GS alpha IND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGS alpha +, contained the coding sequence of GS alpha under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc- cells, and the resulting cells, S49 GS alpha CST cells, expressed GS alpha at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 GS alpha CST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down-regulated the density of beta-adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc- or GS alpha IND cells. When GS alpha CST cells were exposed to 50 nM pindolol for 12 h, the density of beta-adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that GS alpha is necessary to restore the ability of pindolol to down-regulate beta-adrenergic receptors in S49 cyc- cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.     

α2-Adrenoceptor Subtypes Identified by [3H]RX821002 Binding in the Human Brain: The Agonist Guanoxabenz Does Not Discriminate Different Forms of the Predominant α2A Subtype

Abstract: Competition [³H]RX821002 ([³H]2-methoxyidazoxan) binding experiments with α₂-adrenoceptor subtype-specific antagonists—BRL 44408 (α_2A selective), ARC 239 (α_2B selective), and others—were performed to delineate through rigorous computer modeling receptor subtypes in the postmortem human brain. In the hippocampus, hypothalamus, cerebellum, and brainstem the whole population of α₂-adrenoceptors appears to belong to the α_2A subtype (100%; B_max = 34–90 fmol/mg of protein). In the frontal cortex, the predominant receptor was the α_2A subtype (87%; B_max = 53 fmol/mg of protein), although a small population of the α_2B/C subtype (13%; B_max = 8 fmol/mg of protein) was also detected. In the caudate nucleus, a mixed population of α_2A (64%; B_max = 9 fmol/mg of protein) and α_2B/C (36%; B_max = 5 fmol/mg of protein) subtypes was detected. In the cortex and caudate and in the presence of ARC 239 (to mask the α_2B/C-adrenoceptors), competition experiments with the agonist guanoxabenz clearly modeled the high- and low-affinity states of the α_2A subtype. In the presence of ARC 239 and the GTP analogue guanylyl-5′-imidodiphosphate together with NaCl and EDTA (to eliminate the high-affinity α_2A-adrenoceptor) guanoxabenz only recognized the low-affinity α_2A-adrenoceptor. The results indicate that in the human brain the predominant α₂-adrenoceptor is of the α_2A subtype and that this functionally relevant receptor subtype is not heterogeneous in nature.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

Prenatal Diazepam Exposure Affects β-Adrenergic Receptors in Brain Regions of Adult Rat Offspring

The postnatal development of [3H]dihydroalprenolol binding to beta-adrenergic receptors has been studied in frontal cortex, cerebellum, striatum, and hypothalamus of the rat after prenatal and perinatal exposure to diazepam. Dams were injected subcutaneously with single daily doses of 1 mg of diazepam/kg from day 7 to 20 of gestation or from day 15 of gestation to day 6 after birth. Prenatal exposure had no effect on litter size or length of gestation or on the postnatal development of body and brain weights of the progeny. However, a reduced mortality of the pups was observed in relation to vehicle-treated controls until postnatal day 10. Prenatal diazepam administration decreased [3H]dihydroalprenolol binding in frontal cortex, striatum, and hypothalamus but not in cerebellum. This decrease in beta-adrenergic receptor binding was due to a decrease in receptor density rather than in receptor affinity. In contrast, perinatal diazepam exposure led to a transient decrease in [3H]dihydroalprenolol binding limited to the frontal cortex. The permanent reduction in number of beta-adrenergic receptors, which depends on the scaling and duration of the drug application period, points to the necessity of a prolonged evaluation of effects of exposure to psychotropic drugs in early stages of brain development.     

α1-Adrenergic Receptor-Mediated Downregulation of Angiotensin II Receptors in Neuronal Cultures

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.     

Opposite Age-Dependent Changes of α2A-Adrenoceptors and Nonadrenoceptor [3H]Idazoxan Binding Sites (I2-Imidazoline Sites) in the Human Brain: Strong Correlation of I2 with Monoamine Oxidase-B Sites

Abstract: In the postmortem human brain (27 specimens of frontal cortex, Brodmann area 9), the specific binding of the antagonists [³H]RX 821002 (2-methoxyidazoxan) to α_2A-adrenoceptors and that of [³H]idazoxan to l₂-imidazo-line sites (a nonadrenoceptor mitochondrial site) were determined in parallel to study the effect of aging (range, 4–89 years) on both brain proteins. The density of α_2A-adrenoceptors and age were negatively correlated (r=-0.71; p < 0.001). In contrast, the density of l₂-imidazo-line sites was positively correlated with aging (r= 0.59; p < 0.005). The ratio of receptor densities (α_2A/l₂) also showed a marked negative correlation with age (r=-0.76; p < 0.001). In an age-selected group (range, 10–89 years), the density of monoamine oxidase (MAO)-B sites labeled by [³H]Ro 19–6327 (lazabemide) also showed a positive correlation with age (r= 0.80; p < 0.005). In these subjects, the density of l₂-imidazoline sites correlated well with the density of MAO-B sites (r= 0.70; p < 0.005). The ratio of the density of these sites (MAO-B/l₂) did not correlate with the age of the subject at death (r=-0.15). In the human frontal cortex, idazoxan displayed very low affinity (K_i= 89 μM) against the binding of [³H]Ro 19–6327 to MAO-B, which discounted a direct interaction of [³H]idazoxan with the active center of the enzyme and indicated that the l₂-imidazoline site cannot be identified with MAO-B. However, l₂-imidazoline sites and MAO-B show a clear coexpression not only in the human frontal cortex during the process of aging, but also in various brain regions of the human and rat brains. It is suggested that the l₂-imidazoline site has a specific location on glial (astrocyte) cells.     

Solubilization of Active Brain α1-Adrenoceptors by a Zwitterionic Detergent

Solubilization of rat brain alpha 1-adrenoceptors was performed by treatment with 6 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio] - 1 - propanesulfonate). The alpha 1-adrenoceptor antagonist [3H]prazosin was shown to bind reversibly and specifically to the soluble extract obtained after centrifugation at 150,000 X g for 1 h. Separation of the soluble [3H]prazosin-bound complexes was performed by the polyethylene glycol precipitation technique followed by filtration. A Scatchard plot of the concentration-dependent binding curve showed only one class of binding sites, with a high affinity for [3H]prazosin. Affinity of the solubilized receptors for the ligand increased as the CHAPS concentration in the assay medium decreased; the number of binding sites remained unchanged (approximately equal to 70 fmol/mg protein). This corresponds to a 30% recovery of original membrane sites. The solubilized receptors presented the same characteristics of specificity and stereospecificity as membrane alpha 1-adrenoceptors. Moreover, 150 mM NaCl was found to modulate the affinity of epinephrine for the [3H]prazosin-bound soluble complex, as previously described for membrane preparations. Thus, CHAPS appears to be a suitable detergent for solubilizing rat brain alpha 1-adrenoceptors and preserving their functional activities.